Use of 1,3-diphenylprop-2-en-1-one derivatives for treating liver disorders

ABSTRACT

The invention provides 1,3-diphenylprop-2-en-1-one derivatives and pharmaceutical compositions comprising the same for treating liver disorders, in particular those requiring the reduction of plasma level of biochemical markers such as aminotransferases. The 1,3-diphenylprop-2-en-1-one derivatives of General Formula (I) have hepatoprotective properties and can be used in methods for treating liver disorders involving the pathological disruption, inflammation, degeneration, and/or proliferation of liver cells, such as liver fibrosis or fatty liver disease.

This application is a continuation of application Ser. No. 14/933,335(published as US 2016-0051501 A1), filed Nov. 5, 2015 (issued as U.S.Pat. No. 9,585,853 ), which is continuation of application Ser. No.14/523,191 (published as US 2015-0051145 A1), filed Oct. 24, 2014(issued as U.S. Pat. No. 9,221,751), which is a continuation-in-part ofapplication Ser. No. 14/288,482 (issued as U.S. Pat. No. 8,895,619),filed May 28, 2014 (published as US 2014-0309165 A1), which is acontinuation of application Ser. No. 13/511,170, filed May 22, 2012(issued as U.S. Pat. No. 8,772,342), which is a U.S. National Phase ofInternational Application No. PCT/EP2010/068346, filed 26 Nov. 2010,which designated the U.S. and claims priority to European PatentApplication No. 09306146.3, filed 26 Nov. 2009, the entire contents ofeach of which are hereby incorporated by reference.

TECHNICAL FIELD

The invention relates to the use of compounds having hepatoprotectiveeffects in the preparation of pharmaceutical compositions and in methodsfor treating liver disorders.

BACKGROUND

According to the Washington Manual of Medical Therapeutics (31^(st) ed.;2004; Lippincott Williams & Wilkins), liver disorders can be categorizedin different groups of diseases, in particular viral diseases, drug- andalcohol-related liver diseases, immune-mediated liver diseases,metabolic liver diseases, miscellaneous diseases such as non-alcoholicfatty liver disease, and complications of hepatic insufficiency (such asfulminant hepatic failure or hepatocellular carcinoma) and of livertransplantation.

In particular, non-alcoholic fatty liver disease (NAFLD) is a commonhepatic disorder with histological features of alcohol-induced fattyliver disease in individuals who consume little or no alcohol (Yeh M etal., 2007; Marchesini G et al., 2003). NAFLD is due to the abnormalretention of lipids within cells (commonly defined as steatosis), anevent more frequent in liver since this organ is primarily responsibleof lipid metabolism. NAFLD has a spectrum of histological formsincluding hepatic steatosis, and non-alcoholic steatohepatitis (NASH),which is characterized by liver inflammation, steatosis, necrosis andfibrosis due to the disruption of liver cells. Conditions associatedwith NAFLD are varied, and include type 2 diabetes, obesity,dyslipidemia, metabolic syndrome, treatment with hepatotoxic drugs,toxins, infectious agents, or other exogenous causes.

Although NAFLD typically follows a benign, non-progressive clinicalcourse, NASH is a potentially serious condition; as many as 25% ofpatients may progress to advanced fibrosis, cirrhosis and experiencecomplications of portal hypertension, liver failure and hepatocellularcarcinoma, which makes an early and correct assessment mandatory (Yeh Met al, 2007).

Hepatic imaging systems are useful to evaluate also liver structure andpresence of steatosis. However, liver biopsy remains the gold standardfor evaluating liver fibrosis, but this method of analysis could not bedone for every single study due to its invasiveness. Non invasiveevaluation of liver biochemistry and metabolism is often used to defineliver diseases, such as in NAFLD and NASH (Gressner A et al., 2009;Vuppalanchi R and Chalasani N, 2009). By using plasma, high level ofenzymes such as Alanine aminotransferase (ALAT), Aspartateaminotransfersase (ASAT), Alkaline Phosphatase (AP), and/or GammaGlutamyl Transpeptidase (GGT), as well as the presence of other proteinsof liver origin (including haptoglobin, total bilirubin,alpha-2-microglobulin, Resistin, cleaved or intact cytokeratin-18) arecommonly measured in addition to serum glucose and insulin resistanceparameters. Since the level of ALAT activity is frequently increased inNASH patients (Angulo P et al, 2002), this criteria is considered as asurrogate marker for assessing liver injury. In fact, reliablenon-invasive methods are not available to correctly diagnose NAFLD orNASH and even the histological features are not always sufficient todistinguish properly NAFLD or NASH from other conditions such asalcoholic liver disease (Yeh M et al., 2007, Vuppalanchi R and ChalasaniN, 2009).

Means for an effective treatment for liver fibrotic diseases, and NAFLDand NASH in particular, are still insufficient. No treatment isestablished for patient with NASH, and several therapeutic options aretested in clinical trial (Vuppalanchi R and Chalasani N. 2009, Dowman J.K et al., 2009). These studies involve the use of many differentfamilies of chemical compounds (fibrates, thiazolidinediones,biguanides, statins, cannabinoids) and therapeutic targets (nuclearreceptors, angiotensin receptors, cannabinoid receptors, HMG-CoAreductase). Recently, studies involving thiazolidinediones(Rosiglitazone and Pioglitazone) have shown that these drugs may improveliver condition but treatment with these drugs is not without undesiredeffects such as higher risks of congestive cardiac failure andosteoporosis, as well as weight gain with psychological effects on thepatient (Dowman J. K et al., 2009; Shiri-Sverdlov R et al., 2006;Neuschwander-Tetri et al., 2003). Clinical trials involving theadministration of cannabinoids have raised the concern ofneuropsychiatric disruption (Vuppanchi R and Chalasani N, 2009). Othertherapies currently ongoing are seeking to assess in NASH drugs asantioxidants but none of these treatments has yet showed convincingresults (Nelson A et al., 2009).

The need for novel therapeutic options for the management of liverdisorders, in particular those involving liver fibrosis and/orsteatosis, is still clear and urgent.

SUMMARY OF INVENTION

A clinical study has surprisingly shown that the treatment of patientswith a 1,3-diphenylprop-2-en-1-one derivative provides a statisticallyrelevant reduction of liver-specific biochemical markers in the plasma,demonstrating the hepatoprotective properties of a family of compoundsthat is defined by means of a General Formula (I).

The present invention provides novel 1,3-diphenylprop-2-en-1-onederivatives of General Formula (I) (said derivatives being elsewherealso referred to as the “compounds”) or pharmaceutical compositionscomprising the same for use in a method for treating liver disorders, inparticular those ones that lead to the increase of plasma level ofbiochemical markers such as aminotransferases. The1,3-diphenylprop-2-en-1-one derivatives of General Formula (I) andpharmaceutical compositions comprising the same have hepatoprotectiveproperties and can be used in methods for treating liver disordersinvolving the pathological disruption, inflammation, degeneration,and/or proliferation of liver cells, such as liver fibrosis, fatty liverdisease and non-alcoholic steatohepatitis.

Further objects of the present invention, including specific generalformulas of the compounds of interest, are provided in the DetailedDescription.

DESCRIPTION OF THE FIGURES

Abbreviations Used in the Figures and in the Text:

-   -   ALAT=alanine aminotransferase    -   CCL5=chemokine (C-C motif) ligand 5    -   Col1a1=collagen, type I, alpha 1    -   Cpd 1=compound 1 of WO2007/147879    -   Cpd 29=compound 29 of WO2004/005233    -   Ctrl=control or vehicle    -   Feno=Fenofibrate    -   HDL=High Density Lipoprotein    -   LDL=Low Density Lipoprotein    -   NAFLD=Non-alcoholic fatty liver disease    -   NASH=Non-alcoholic steatohepatitis    -   PPAR=Peroxisome Proliferator Activated Receptor    -   Rosi=Rosiglitazone    -   RT-PCR=Reverse Transcription Polymerase Chain Reaction    -   TGFβ=Transforming Growth Factor beta    -   TNFα=Tumor Necrosis Factor alpha

FIG. 1: Structure of Exemplary Compounds of General Formula (I)

Exemplary compounds of the General Formula (I) are grouped according tothe more specific definitions of General Formula (II) (Panel A), ofGeneral Formula (IV) (Panel B), and of General Formula (V) (Panel C).

FIG. 2 In Vivo Evaluation, in the Ob/Ob Mice, of the Anti-InflammatoryProperties of Compounds of the General Formula (I)

The compounds of the General Formula (I) were tested in a murine modelof type II diabetes, the ob/ob mice. Mice were daily orally treated withthe Compound 29 of WO2004/005233 at two different doses (10 and 30mg/kg/day) and with the prototypical PPARalpha- and PPARgamma-specificreference compounds (Fenofibrate at 100 mg/kg/day and Rosiglitazone at10 mg/kg/day respectively). After 26 days of treatment, animals weresacrificed and plasma samples and livers were harvested. Hepaticexpression of genes that are known to be implicated in the liverinflammation process was evaluated and plasmatic levels of ALAT weremeasured (Panels A-C). Statistical analysis was performed using unpairedT-test with three p values that defines statistical relevance (* meansp<0.05; ** means p<0.01; *** means p<0.001).

FIG. 3: In Vivo Evaluation, in the hApoE2 KI Mice, of theAnti-Inflammatory and Anti-Fibrotic Properties of Compounds of theGeneral Formula (I)

The compounds of the General Formula (I) were tested in vivo in a highfat diet mice model. Dyslipidemic “humanized” ApoE2 knock-in mice(hApoE2 KI) were fed a Western diet and treated during 12 weeks. Thecompounds of interest, including the Compound 29 of WO2004/005233 at 0.3mg/kg/day and the Fenofibrate at 100 mg/kg/day (used as a referencecompound) were incorporated into the diet. At the end of the protocol,animals were sacrificed, livers were harvested and the hepaticexpression of genes that are known to be implicated in the liverinflammation and the fibrosis processes were evaluated by quantitativeRT-PCR (Panels A-D). Statistical analysis was performed as indicated forFIG. 1.

FIG. 4. In Vivo Evaluation, in the hApoE2 KI and in the hApE2KI/PPARalpha KO Mice, of the Anti-Inflammatory and Anti-SteatoticProperties of Compounds of the General Formula (I)

The compounds of the General Formula (I) were tested in vivo in a highfat diet mice model. Dyslipidemic “humanized” hApoE2 KI deficient forPPARalpha were fed a Western diet and treated during 6 weeks. Thecompounds of interest, including the Compound 29 of WO2004/005233 at 30mg/kg/day and the Compound 1 of WO2007/147879 at 30 mg/kg/day wereorally administrated by gavage. At the end of the protocol, animals weresacrificed, livers were harvested and the hepatic expression of relevantgenes implicated in the liver inflammation and the fibrosis processeswere evaluated by quantitative RT-PCR. In parallel, liver triglyceridescontents were evaluated (Panels A-D). Statistical analysis was performedas indicated for FIG. 1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel therapeutic uses and methods ofadministration of 1,3-diphenylprop-2-en-1-one derivatives of GeneralFormula (I) and pharmaceutical compositions comprising the same fortreating liver disorders. Specific 1,3-diphenylprop-2-en-1-onederivatives that are substituted on both phenyl groups can be definedaccording to the Examples as being useful for treating liver disorders,since such compounds decrease in a surprising manner specific markers ofliver inflammation as well as of the disruption, degeneration, and/orproliferation of liver cells in human subjects and animal models, andthus they can provide an hepatoprotective effect.

The compounds to be used and administered according to the invention andcomprised in the compositions according to the invention have thefollowing General Formula (I):

-   -   in which:    -   X1 represents a halogen, a R1, or G1-R1 group;    -   A represents a CH═CH or a CH2-CH2 group;    -   X2 represents a G2-R2 group;    -   G1 and G2, identical or different, represent an atom of oxygen        or sulfur;    -   R1 represents a hydrogen atom, an unsubstituted alkyl group, an        aryl group or an alkyl group that is substituted by one or more        halogen atoms, an alkoxy or an alkylthio group, cycloalkyl        groups, cycloalkylthio groups or heterocyclic groups;    -   R2 represents an alkyl group substituted by at least a —COOR3        group, wherein R3 represents a hydrogen atom, or an alkyl group        that is substituted or not by one or more halogen atoms,        cycloalkyl groups, or heterocyclic groups.    -   R4 and R5, identical or different, representing an alkyl group        that is substituted or not by one or more halogen atoms,        cycloalkyl groups, heterocyclic groups.

In a particular embodiment, compounds of General Formula (I) aresubstituted by at least an alkyloxy group or an alkylthio group in X1and X2 positions. Moreover, the derivatives can be in the form ofsubstituted 1,3-diphenylpropanones that are obtained by reduction of thecorresponding 1,3-diphenylprop-2-en-1-one derivatives.

In a particular embodiment, X1 is a G1-R1 group, and more preferably G1is a sulfur atom and R1 is a linear or branched alkyl group that issubstituted or not by one or more halogen atoms, cycloalkyl groups,heterocyclic groups. Even more preferably, X1 is an alkylthio group thatcomprises an alkyl group that is linear or branched, having from one toseven carbon atoms that is substituted or not by one or more halogenatoms. In a preferred embodiment, X1 is a methylthio group.

In a particular embodiment, X2 is a G2-R2 group wherein G2 is a oxygenatom and R2 is an alkyl group substituted by a —COOR3 group, wherein R3represents a hydrogen atom or an unsubstituted linear or branched alkylgroup having from one to seven carbon atoms, and more preferably fromone to four carbon atoms. In a preferred embodiment, both R4 and R5represent methyl groups.

Furthermore, R4 and R5, identical or different, are preferablyunsubstituted linear branched, alkyl groups having from one to sevencarbon atoms, and more preferably from one to four carbon atoms.

In the context of the present invention, the term “alkyl” refers to asaturated hydrocarbon radical that is linear or branched, havingpreferably from one to twenty-four, and even more preferably from one toseven carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, tertiobutyl, sec-butyl, pentyl, neopentyl, or n-hexyl.

The term “alkyloxy” refers to an alkyl group that is linked to theremainder of the compound by an oxygen atom.

The term “alkylthio” refers to an alkyl group that is linked to theremainder of the compound by a sulfur atom (thioether bond).

The term “cycloalkyl” designates an alkyl group that forms one cyclehaving preferably from three to fourteen carbon atoms, and morepreferably three to eight carbon atoms, such as cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

The term “cycloalkylthio” refers to a cycloalkyl group that is linked tothe remainder of the compound by a sulfur atom (thioether bond).

The term “aryl” designates an aromatic group, substituted or not havingpreferably from six to fourteen carbon atoms such as phenyl, a-naphtyl,b-naphtyl, biphenyl, or anthracenyl.

The term “heterocyclic” refers to a heterocycloalkyl group or aheteroaryl group.

The term “heterocycloalkyl” group refers to a cycloalkyl as indicatedabove that further comprises one or several heteroatoms selected amongnitrogen, oxygen or sulfur. They generally comprise from four tofourteen carbon atoms, such as morpholinyl, piperidinyl,tetrahydropyranyl, dithiolanyl.

The term “heteroaryl” refers to an aryl group as indicated above thatfurther comprises one or several heteroatoms selected among nitrogen,oxygen or sulfur. They generally comprise from four to fourteen carbonatoms, such as furanyl, thiophenyl, pyridinyl, pyrimidinyl, quinoleinyl,isoquinoleinyl.

By halogen atom, an atom of bromine, chlorine, fluorine or iodine isunderstood.

Different families of 1,3-diphenylprop-2-en-1-one derivatives and1,3-diphenylpropanones that are substituted on both phenyl groups can befound in the prior art (WO2003/037315, WO2001/046110. JP2006-303800,JP04-202129). However, none of these documents shows that specifichepatoprotective effects are associated to compounds as defined inGeneral Formula (I).

The structure, synthesis, and some activities of compounds that areencompassed by General Formula (I) have been disclosed in a series ofpatent applications (WO2004/005243, WO2004/005233, WO2005/005369,US20070032543, WO2005/073184, WO2007/147879, and WO2007/147880) that donot disclose the use of such compounds in methods for treating liverdisorders.

Specific 1,3-diphenylprop-2-en-1-one derivatives of General Formula (I)that can be used in the present invention and that can be comprised incompositions according to the invention can be selected from thosedisclosed in WO2004/005243 and WO2004/005233, and in particular:

-   1-[4-chlorophenyl]-3-[3,5-dimethyl-4-isopropyloxycarbonyldimethylmethyloxyphenyl]prop-2-en-1-one,    described as compound 15;-   1-[4-chlorophenyl]-3-[3,5-dimethyl-4-tertbutyloxycarbonyldimethylmethyloxyphenyl]prop-2-en-1-one,    described as compound 16;-   1-[4-chlorophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one    as compound 17;-   1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-tertbutyloxycarbonyldimethylmethyloxyphenyl]prop-2-en-1-one,    described as compound 27;-   1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-isopropyloxycarbonyldimethylmethyloxyphenyl]prop-2-en-1-one,    described as compound 28;-   1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one,    described as compound 29;-   1-[4-hexyloxyphenyl]-3-[3,5-dimethyl-4-tertbutyloxycarbonyldimethylmethyloxyphenyl]prop-2-en-1-one,    described as compound 32;-   1-[4-hexyloxyphenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one,    described as compound 33;-   1-[4-heptylphenyl]-3-[3,5-dimethyl-4-tertbutyloxycarbonyldimethylymethyloxyphenyl]prop-2-en-1-one,    described as compound 38;-   1-[4-heptylphenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one,    described as compound 39;-   1-[4-bromophenyl]-3-[3,5-dimethyl-4-tertbutyloxycarbonyldimethylmethyloxyphenyl]prop-2-en-1-one,    described as compound 40;-   1-[4-bromophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one,    described as compound 41.

In a further embodiment of the invention, the compounds as disclosed inWO2004/005243 and WO2004/005233 (herein referred to as compounds ofGeneral Formula (II)) that can be used and administered and that can becomprised in compositions according to the invention have the followingGeneral Formula (I):

-   -   in which:    -   X1 represents a halogen, a R1, or a G1-R1 group;    -   A represents a CH═CH group;    -   X2 represents a G2-R2 group;    -   G1 and G2, identical or different, represent an atom of oxygen        or sulfur;    -   R1 represents an alkyl or cydoalkyl group having from one to        seven carbon atoms, in particular, the alkyl or cycloalkyl group        being substituted or not by one or more halogen atoms;    -   R2 represents an alkyl group substituted by a —COOR3 group,        wherein R3 represents a hydrogen atom or an alkyl group having        from one to four carbon atoms.    -   R4 and R5 represent an alkyl group having from one to four        carbon atoms.

WO2005/005369 and US20070032543 also disclose the structure andalternative process for the synthesis of compounds according to GeneralFormula (I) as well as to General Formula (II), in particular:

-   1-[4-trifluoromethylphenyl]-3-[3,5-dimethyl-4-tertbutyloxycarbonyldimethylmethyloxyphenyl]prop-2-en-1-one    (compound 57 of US20070032543)-   1-[4-trifluoromethylphenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyoxyphenyl]prop-2-en-1-one    (compound 58 of US20070032543)-   1-[4-trifluoromethyloxyphenyl]-3-[3,5-dimethyl-4-tertbutyycarbonydimethylmethyloxyphenyl]prop-2-en-1-one    (compound 61 of US20070032543)-   1-[4-trifluoromethyloxyphenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyoxyphenyl]prop-2-en-1-one    (compound 62 of US20070032543).

Additional examples of compounds to be used and administered and thatcan be comprised in compositions according to the invention can beselected from those disclosed in WO2005/073184, and in particular

-   1-(4-(Pentylthioethyloxy)phenyl)-3-(4-tert-butyloxycarbonyldimethylmethyloxy-3,5-dimethylphenyl)prop-2-en-1-one,    described as compound 1;-   1-(4-(Pentylthioethyloxy)phenyl)-3-(4-carboxydimethylmethyloxy-3,5-dimethylphenyl)prop-2-en-1-one,    described as compound 2;-   1-(4-((R,S)-5-[1,2]dithiolan-3-ylpentyloxy)phenyl)-3-(4-tert-butyloxycarbonydimethylmethyloxy-3,5-dimethylphenyl)prop-2-en-1-one,    described as compound 5;-   1-(4-((R,S)-5-[1,2]dithiolan-3-ylpentyloxy)phenyl)-3-(4-carboxydimethylmethyloxy-3,5-dimethylphenyl)prop-2-en-1-one,    described as compound 6;-   1-(4-Cyclohexylethyloxyphenyl)-3-(4-tert-butyloxycarbonyldimethylmethyloxy-3,5-dimethylphenyl)prop-2-en-1-one,    described as compound 10;-   1-(4-Cyclohexylethyloxyphenyl)-3-(4-carboxydimethylmethyloxy-3,5-dimethylphenyl)prop-2-en-1-one,    described as compound 11;-   1-(4-Cyclohexylthioethyloxyphenyl)-3-(4-tert-butyoxycarbonyldimethylmethyoxy-3,5-dimethylphenyl)prop-2-en-1-one,    described as compound 22;-   1-(4-Cyclohexylthioethyloxyphenyl)-3-(4-carboxydimethylmethyloxy-3,5-dimethylphenyl)prop-2-en-1-one,    described as compound 23:-   1-(4-Phenyloxyphenyl)-3-(4-tert-butyloxycarbonyldimethylmethyoxy-3,5-dimethylphenyl)prop-2-en-1-one,    described as compound 32;-   1-(4-Phenyloxyphenyl)-3-(4-carboxydimethylmethyloxy-3,5-dimethylphenyl)prop-2-en-1-one,    described as compound 33.

In a further embodiment of the invention, the compounds as disclosed inWO2005/073184 (herein referred to as compounds of General Formula (III))that can be used and administered, and that can be comprised incompositions according to the invention have the following GeneralFormula (I):

-   -   in which:    -   X1 represents a G1-R1 group;    -   A represents a CH═CH group;    -   X2 represents a G2-R2 group;    -   G1 and G2 represent an atom of oxygen;    -   R1 represents an cycloalkyl, an aryl or an alkyl group that is        substituted or not by one or more alkylthio, cycloalkyl,        cycloalkylthio groups or heterocycloalkyl groups or an alkylthio        group;    -   R2 represents an alkyl group substituted by at least a —COOR3        group, wherein R3 represents a hydrogen atom or an alkyl group        having from one to four carbon atoms;    -   R4 and R5 represent an alkyl group having from one to four        carbon atoms.

Additional examples of compounds used and administered according to theinvention and that can be comprised in compositions according to theinvention can be selected from those disclosed in WO2004/005243,WO2004/005233, WO2005/005369, US20070032543 or WO2005/073184, andreduced in the form of the corresponding substituted1,3-diphenylpropanones.

Accordingly, compounds that can be used and administered according tothe invention and that can be comprised in compositions according to theinvention can be selected from those disclosed in WO2007/147879, and inparticular:

-   2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-propyl]phenoxy]-2-methylpropanoic    acid, described as compound 1;-   2-[2,6-dimethyl-4-[3-[4-(methoxy)phenyl]-3-oxo-propyl]phenoxy]-2-methylpropanoic    acid, described as compound 6;-   2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-propyl]phenoxy]ethanoic    acid, described as compound 7;-   2-[2,6-dimethyl-4-[3-[4-(propyloxy)phenyl]-3-oxo-propyl]phenoxy]-2-methylpropanoic    acid, described as compound 8;-   2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-propyl]phenoxy]-2-methyl    propanoic acid isopropyl ester, described as compound 13.

In a further embodiment of the invention, the compounds as disclosed inWO2007/147879 (herein referred to as compounds of General Formula (IV))that can be used and administered and that can be comprised incompositions according to the invention have the following GeneralFormula (I):

-   -   in which:    -   X1 represents a R1 or a G1-R1 group;    -   A represents a CH2-CH2 group;    -   X2 represents a G2-R2 group;    -   G1 represents an atom of oxygen or sulfur and G2 represents an        atom of oxygen;    -   R1 represents an alkyl or cycloalkyl group having from one to        seven carbon atoms;    -   R2 represents an alkyl group substituted by at least a —COOR3        group, wherein R3 represents a hydrogen atom or an alkyl group        having from one to four carbon atoms;    -   R4 and R5 represent an alkyl group having from one to four        carbon atoms.

Similarly to WO2007/147879, WO2007/147880 discloses compounds that canbe comprised in compositions according to the invention that correspondto reduced, substituted 1,3-diphenylpropanones derivatives of compoundsthat were disclosed in WO2004/005243, WO2004/005233, WO2005/005369,US20070032543, or WO2005/073184, and in particular:

-   2-[2,6-dimethyl-4-[3-[4-(trifluoromethyloxy)phenyl]-3-oxo-propyl]phenoxy]-2-methyl-propanoic    acid, described as compound 1;-   2-[2,6-dimethyl-4-[3-[4-(trifluoromethylthio)phenyl]-3-oxo-propyl]phenoxy]-2-methyl    propanoic acid, described as compound 2;-   2-[2,6-dimethyl-4-[3-[4-bromophenyl]-3-oxo-propyl]phenoxy]-2-methylpropanoic    acid, described as compound 3;-   2-[2,6-dimethyl-4-[3-[4-(trifluoromethyl)phenyl]-3-oxo-propyl]phenoxy]-2-methylpropanoic    acid, described as compound 4;-   2-[2,6-dimethyl-4-[3-[4-(3,3,3-trifluoropropyloxy)phenyl]-3-oxo-propyl]phenoxy]-2-methyl    propanoic acid, described as compound 11;-   2-(2,6-dimethyl-4-(3-oxo-3-(4-(2,2,2-trifluoroethoxy)phenyl)propyl)phenoxy)-2-methylpropanoic    acid, described as compound 12;-   2-(2,6-dimethyl-4-(3-oxo-3-(4-(2,2,2-trifluoroethylthio)phenyl)propyl)phenoxy)-2-methyl    propanoic acid, described as compound 13-   2-(2,6-dimethyl-4-(3-oxo-3-(4-(trifluoromethoxy)phenyl)propyl)phenoxy)propanoic    acid, described as compound 29;-   4-(2,6-dimethyl-4-(3-oxo-3-(4-(trifluoromethoxy)phenyl)propyl)phenoxy)-2,2-dimethyl    butanoic acid, described as compound 34;-   2-(2,6-dimethyl-4-(3-oxo-3-(4-(trifluoromethoxy)phenyl)propyl)phenoxy)-2-methyl    propanoic acid tertiobutyl ester, described as compound 35;-   2-(2,6-dimethyl-4-(3-oxo-3-(4-(trifluoromethoxy)phenyl)propyl)phenoxy)-2-methyl    propanoic isopropyl ester, described as compound 36;-   2,2-difluoro-2-(2,6-dimethyl-4-(3-oxo-3-(4-(trifluoromethoxy)phenyl)propyl)phenoxy)acetic    acid, described as compound 37.

In a further embodiment of the invention, the compounds as disclosed inWO2007/147880 (herein referred to as compounds according to GeneralFormula (V)) that can be used and administered and that can be comprisedin compositions according to the invention have the following GeneralFormula (I):

-   -   in which:    -   X1 represents a halogen atom or a R1 or G1-R1 group;    -   A represents a CH2-CH2 group;    -   X2 represents a G2-R2 group;    -   G1 represents an atom of oxygen or sulfur and G2 represents an        atom of oxygen;    -   R1 represents an alkyl or cycloalkyl group that is substituted        by one or more halogen atoms;    -   R2 represents an alkyl group substituted or not by one or more        halogen atoms and substituted by at least a —COOR3 group,        wherein R3 represents a hydrogen atom or an alkyl group having        from one to four carbon atoms.    -   R4 and R5 represent an alkyl group having from one to four        carbon atoms.

The compounds that can be most preferably used and administeredaccording to the invention and comprised in compositions according tothe invention are those defined according to General Formula (II),General Formula (IV) or General Formula (V), and in particular

-   1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one    (compound 29 of WO2004/005233);-   1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-isopropyloxycarbonyldimethylmethyloxyphenyl]prop-2-en-1-one    (compound 28 of WO2004/005233);-   1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-tertbutyloxycarbonyldimethylmethyloxyphenyl]prop-2-en-1-one    (compound 27 of WO2004/005233);-   1-[4-trifluoromethylphenyl]-3-[3,5-dimethyl-4-tertbutyloxycarbonyldimethylmethyloxyphenyl]prop-2-en-1-one    (compound 57 of US20070032543)-   1-[4-trifluoromethylphenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyioxyphenyl]prop-2-en-1-one    (compound 58 of US20070032543)-   1-[4-trifluoromethyloxyphenyl]-3-[3,5-dimethyl-4-tertbutyloxycarbonyldimethylmethyloxyphenyl]prop-2-en-1-one    (compound 61 of US20070032543)-   1-[4-trifluoromethyloxyphenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one    (compound 62 of US20070032543)-   2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-propyl]phenoxy]-2-methylpropanoic    acid (compound 1 of WO2007/147879);-   2-[2,6-dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-propyl]phenoxy]-2-methyl-propanoic    acid isopropyl ester (compound 13 of WO2007/147879);-   2-[2,6-dimethyl-4-[3-[4-(trifluoromethyloxy)phenyl]-3-oxo-propyl]phenoxy]-2-methylpropanoic    acid (compound 1 of WO 2007147880);-   2-[2,6-dimethyl-4-[3-[4-(trifluoromethylthio)phenyl]-3-oxo-propyl]phenoxy]-2-methylpropanoic    acid (compound 2 of WO 2007147880);-   2-(2,6-dimethyl-4-(3-oxo-3-(4-(trifluoromethyloxy)phenyl)propyl)phenoxy)-2-methylpropanoic    acid tert-butyl ester (compound 35 of WO 2007147880).

Other compounds that may be used and administered according to theinvention and comprised in compositions according to the invention arein particular:

-   1-[4-methylthiophenyl]-3-[3,5-diisopropyl-4-carboxymethyloxyphenyl]prop-2-en-1-one    (compound 74 of US 2007/0032543);-   1-(4-Cyclohexylethyloxyphenyl)-3-(4-carboxydimethylmethyloxy-3,5-dimethylphenyl)prop-2-en-1-one    (compound 11 of WO 2005/073184);-   1-[4-isopropylphenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one    (compound 56 of US 2007/0032543);-   1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxymethyloxyphenyl]prop-2-en-1-one    (compound 72 of US 2007/0032543);-   1-[4-hexyloxyphenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one    (compound 33 of WO 2004/005233);-   1-[4-hexylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one    (compound 84 of US 2007/0032543);-   1-[4-trifluoromethylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one    (compound 54 of US 2007/0032543);-   1-[4-methyloxyphenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one    (compound 80 of US 2007/0032543);-   1-(4-Phenyloxyphenyl)-3-(4-carboxydimethylmethyloxy-3,5-dimethylphenyl)prop-2-en-1-one    (compound 33 of WO 2005/073184);-   1-[4-chlorophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one    (compound 17 of WO 2004/005233);-   1-[4-bromophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one    (compound 41 WO 2004/005233);-   1-[4-trifluoromethylphenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one    (compound 58 of US 2007/0032543).

The present invention provides specific uses of compounds of GeneralFormula (I) and related pharmaceutical compositions comprising the same.The compound may or may or not be in the form of a pharmaceuticallyacceptable salt and is used in a therapeutically effective amount fortreating liver disorders Any compound that is defined according toGeneral Formula (II), General Formula (III), General Formula (IV), orGeneral Formula (V), these formulae being encompassed by General Formula(I), can be used in the present invention for treating liver disorders,in particular in the form of a pharmaceutical composition that comprisessaid compound.

The invention also provides a method for treating liver disorderscomprising the administration to a subject in need thereof of a compoundof General Formula (I) in which:

-   -   X1 represents a halogen, a R1 or G1-R1 group;    -   A represents a CH═CH or a CH2-CH2 group;    -   X2 represents a G2-R2 group;    -   G1 and G2, identical or different, represent an atom of oxygen        or sulfur;    -   R1 represents a hydrogen atom, an unsubstituted alkyl group, an        aryl group or an alkyl group that is substituted by one or more        halogen atoms, an alkoxy or an alkylthio group, cycloalkyl        groups, cycloalkylthio groups or heterocyclic groups;    -   R2 represents an alkyl group substituted by at least a —COOR3        group, wherein R3 represents a hydrogen atom, or an alkyl group        that is substituted or not by one or more halogen atoms,        cycloalkyl groups, or heterocylic groups.    -   R4 and R5, identical or different, representing an alkyl group        that is substituted or not by one or more halogen atoms,        cycloalkyl groups, heterocylic groups.

Compositions comprising compounds wherein X1, X2, A, G1, G2, R1, R2, R3,R4, and R5 are defined according to General Formula (II), GeneralFormula (III), General Formula (IV), or General Formula (V) can also beused to carry out the method for treating liver disorders.

The term “liver disorder” includes any disorder affecting the liver, andin particular any acute or chronic liver disease that involves thepathological disruption, inflammation, degeneration, and/orproliferation of liver cells. In particular, the liver disorder is liverfibrosis, liver cirrhosis, or any other liver disease in which the levelin the plasma of some markers of hepatocellular injury, alteration ornecrosis, is elevated when compared to normal plasma levels. Thesebiochemical markers associated to liver activity and status can beselected among those disclosed in the literature and in particularAlanine aminotransferase (ALAT), Aspartate aminotransfersase (ASAT),Alkaline Phosphatase (AP), Gamma Glutamyl transpeptidase (GGT),Cytokeratin-18 (CK-18) or Resistin. In a particular embodiment, theliver disorder is a fatty liver disease in which the elevation of one ormore of these markers is associated to a more or less significantsteatosis in the liver, as it can be confirmed by a liver biopsy. Anon-exhaustive list of fatty liver diseases includes NAFLD, NASH, andfatty liver disease associated to disorders such as hepatitis ormetabolic syndrome (obesity, insulin resistance, hypertriglyceridemia,and the like).

The term “Hepatoprotection” or “Hepatoprotective” refers to the abilityof a compound to reduce, reverse or prevent damage to the liver, inparticular by reducing, reversing or preventing the pathologicaldisruption, inflammation, degeneration, and/or proliferation of livercells such as hepatocytes.

The term “treatment” or “treating” refers to therapy, prevention andprophylaxis of a disorder, in particular of a liver disorder. Thetreatment involves the administration of a compound or pharmaceuticalcomposition to patient having a declared disorder to cure, delay, orslow down the progress, thus improving the condition of patients. Thetreatment may be also administered to healthy subjects that are at riskof developing a liver disorder.

Within the context of the invention, the term “subject” means a mammaland more particularly a human. The subjects to be treated according tothe invention can be appropriately selected on the basis of severalcriteria associated to the liver disorder such as previous drugtreatments, associated pathologies, genotype, exposure to risk factors,viral infection, as well as any other relevant biomarker that can beevaluated by means of immunological, biochemical, enzymatic, chemical,or nucleic acid detection method. In a particular embodiment, thesubject is an overweighed patient (in particular an overweighedprediabetic or diabetic patient) or obese patient suffering fromatherogenic dyslipidemia. Indeed, these patients are at risk ofdeveloping a liver disorder, in particular NAFLD or NASH. The inventorshave shown that compounds as defined above have a beneficial effect onhepatic functions of such patients.

The compounds of General Formula (I) may contain one or severalasymmetrical centers. When an enantiomerically pure (or enriched)compound is desired, it can be obtained either by purification of thefinal product or chiral intermediates, or by asymmetrical synthesisfollowing the typical methods known by one of ordinary skill in the art(for example, by using reactives and chiral catalysts). Some of thesecompounds can have different stable tautomeric forms. This inventionincludes the use of stereoisomers (diastereoisomers, enantiomers), pureor mixed, as well as racemic mixtures and geometrical isomers ofcompounds of General Formula (I).

The compounds of General Formula (I) can be formulated as“pharmaceutically acceptable” salts, being slightly- or non-toxic saltsobtained from organic or inorganic bases or acids of compounds ofGeneral Formula (I). These salts can be obtained during the finalpurification step of the compound or by incorporating the salt into thepreviously purified compound.

The pharmaceutical compositions comprising a compound of General Formula(I) for the treatment of liver disorders can comprise one or severalexcipients or vehicles, acceptable within a pharmaceutical context (e.g.saline solutions, physiological solutions, isotonic solutions, etc.,compatible with pharmaceutical usage and well-known by one of ordinaryskill in the art). These compositions can comprise one or several agentsor vehicles chosen among dispersants, solubilisers, stabilisers,preservatives, etc. Agents or vehicles useful for these formulations(liquid and/or injectable and/or solid) are particularlymethylcellulose, hydroxymethylcellulose, carboxymethylcellulose,polysorbate 80, mannitol, gelatin, lactose, vegetable oils, acacia,liposomes, etc. These compositions can be formulated in the form ofinjectable suspensions, gels, oils, pills, suppositories, powders, gelcaps, capsules, aerosols, etc., eventually by means of galenic forms ordevices assuring a prolonged and/or slow release. For this kind offormulation, agents such as cellulose, carbonates or starches canadvantageously be used.

The compounds of General Formula (I) should be administered in aneffective quantity of a compound by using a pharmaceutical compositionas above-defined. Within the context of the invention, the term “aneffective quantity” refers to an amount of the compound sufficient toproduce the desired therapeutic result.

The compounds of General Formula (I) can be administered in differentways and in different forms that allow administering said compounds in atherapeutically effective amount. Thus, for example, they can beadministered in a systematic way, per os, parenterally, by inhalation,or by injection, such as for example intravenously, by intra-muscularroute, by subcutaneous route, by transdermal route, by intra-arterialroute, etc. Oral administration is the preferential route ofadministration for pharmaceutical compositions comprising a compound ofGeneral Formula (I) for the treatment of liver disorders.

The frequency and/or dose relative to the administration can be adaptedby one of ordinary skill in the art, in function of the patient, thepathology, the form of administration, etc. Typically, the compounds ofGeneral Formula (I) can be administered for the treatment of liverdisorders at doses varying between 0.01 mg and 1 g per administration,preferentially from 1 mg to 100 mg per administration. Administrationcan be performed daily or even several times per day, if necessary.

The compounds and compositions of the invention can be advantageouslyadministered in combination with other therapeutic agents, currentlyavailable in the market or in development for the treatment of metabolicand/or liver disorders, such as metformin, insulin, thiazolidinediones,glitazones, statins, inhibitors of cholesterol and/or other lipidlowering drugs.

In a further embodiment, the present invention provides methods oftreating liver disorders comprising the administration of a compound ofGeneral Formula (I), in particular in the form of pharmaceuticalcompositions containing these compounds. Such methods may comprise theadministration of any compound that is defined according to GeneralFormula (II), General Formula (III), General Formula (IV), or GeneralFormula (V).

The compounds and compositions of the invention provide advantageoustherapeutic tool for the treatment of liver disorders, and in particularfatty liver diseases including NAFLD and NASH, due to thehepatoprotective effects of the compounds of General Formula (I). Inparticular, these compounds can be selected amongst those in which X1,X2, A, G1, G2, R1, R2, R3, R4, and R5 are defined according to GeneralFormula (II), General Formula (III), General Formula (IV), or GeneralFormula (V). A further object of the present invention relates to acompound of General formula (I) as described above, and in particular ofGeneral Formula (II), (III), (IV) and (V), for use in a method oftreating liver disorders. In a particular embodiment, specific liverdisorders intended to be treated are those described above such as liverfibrosis or a fatty liver disease. In yet another embodiment, thecompounds for use in said methods are those specifically describedabove.

In general, the liver-specific properties of compounds of GeneralFormula (I) can be evaluated in specific patient populations presentinga liver disorder such as NAFLD and/or NASH at inclusion. For example, adouble blind, placebo-controlled and randomized study can evaluate theefficacy of oral administration of the compound (at the dose of 80mg/day or more) during 3-12 months in subjects that have been diagnosedfor NAFLD (steatosis only) and/or NASH (steatosis and fibrosis) andpresent elevated aminotransferases levels. Any statistically relevantimprovement on main biochemical parameters (such as reduction ofaminotransferases, GGT, and/or Cytokeratin-18 levels and/or reduction ofResistin levels), on volume of hepatic steatosis measured by imagingtechnique or on histological features of liver biopsies (measurement ofsteatosis, liver inflammation and fibrosis) can be regularly assessed inthese patients during the study (on a monthly or more frequent basis).Additional parameters such as total/LDL-/HDL-cholesterol, hemodynamicparameters, Body Mass Index, insulin resistance, markers of inflammatoryor oxidative stress, plasma insulin and glucose, markers of renalfunction in urine, hepatic imaging by MRI, and/or histomorphology inliver biopsies can be also measured during the study and/or at the endof the study for completing the efficacy profile of compounds fortreating of liver disorders.

All references cited herein are fully incorporated by reference in theirentirety. Having now fully described the invention, it will beunderstood by those of ordinary skill in the art that the invention maybe practiced within a wide and equivalent range of conditions,parameters and the like, without affecting the spirit or scope of theinvention or any embodiment thereof. Several other advantages of theinvention will rise in the reading of the following examples; theyshould be considered as illustrative data and not as limitative ones.

EXAMPLES Example 1 Effects of Compounds of General Formula (I) onLiver-Specific Biochemical Indexes

Materials & Methods

1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-one(Cpd 29 of WO2004/005233) has been formulated as hard shell capsulescontaining 5, 10 or 20 mg of the compound. The compound (80 mg) wasadministered orally once daily for 28 days. The study has been performedin two parallel groups in double-blind conditions: placebo or Cpd 29 ofWO2004/005233.

The tolerability and safety of once-a-day administrations, as well asthe efficacy in improving plasma lipids and glucose homeostasis comparedwith placebo, were evaluated in two pilot trials using relevantbiochemical parameters. The data were used to calculate the percentageof change due to the compound when compared to the placebo after 28 daysof treatment.

Results & Conclusions:

A first pilot, double-blind, placebo controlled, randomized study hasbeen performed in patients suffering from atherogenic dyslipidaemia andabdominal obesity for assessing the tolerability and safety ofonce-a-day administrations of oral doses of Cpd 29 of WO2004/005233 (atthe dose of 80 mg/day), as well as the efficacy on plasma triglyceridesand HDL-cholesterol (primary objectives).

Relative to the placebo group, the therapeutic efficacy of this compoundwas demonstrated with a statistically significant 21% (p<0.01) reductionof plasma triglycerides and a 9% (p<0.01) increase in good cholesterol(HDL-C) level. These metabolic effects were comparable to thosepublished with the fibrates in the same patient population. Furthermore,the compound revealed a remarkable lack of effect on Homocystein (aknown cardiovascular risk factor). The compound showed significanteffects on multiple secondary evaluation criteria including reduction ofliver acute phase inflammation markers such as fibrinogen andhaptoglobin (p<0.01). Effects on biochemical parameters of liverfunction were also measured and the administrations of oral doses of Cpd29 of WO2004/005233 led unexpectedly to a statistically significant 23%reduction of Gamma Glutamyl transpeptidase level (p<0.001) and a 13%reduction of Alanine aminotransferase level (p<0.01).

A second pilot, double-blind, placebo controlled, randomized study hasbeen performed in patients suffering from impaired fasting glucose,impaired glucose tolerance and abdominal obesity for assessing thetolerability and safety of once-a-day administrations of oral doses ofCpd 29 of WO2004/005233 (at the dose of 80 mg/day), as well as theefficacy on glucose and lipid metabolism.

Relative to the placebo group, the therapeutic efficacy of this compoundwas demonstrated with a statistically significant reduction of fastingplasma glucose (−5%, p<0.05), of fasting insulinemia (−25%, p<0.01) andof insulin resistance index, HOMA-IR (−31%, p<0.01). In parallel, Cpd 29of WO2004/005233 reduced plasma triglycerides (−25%, p<0.001) and LDL-Cwhile enhancing HDL-C (+9%, p<0.01). The compound showed significanteffects on multiple secondary evaluation criteria including reduction ofliver acute phase inflammation markers such as haptoglobin (p<0.01).Biochemical parameters on liver function were also calculated and theadministrations of oral doses of Cpd 29 of WO2004/005233 led to astatistically 15% reduction of Gamma Glutamyl transpeptidase level(p<0.01).

These results demonstrated that an oral formulation of a compound ofGeneral Formula (I) not only is well tolerated by patients but haspositive effects on multiple biochemical parameters associated withNAFLD and NASH including liver enzymes, insulin sensitivity, lipidmetabolism, and liver inflammation markers. In particular, Cpd 29 ofWO2004/005233 significantly decreases plasma levels of ALAT and GGT, twocommon specific biomarkers of liver dysfunction which are elevated inpatients suffering from NAFLD and NASH.

Example 2 Animal Models for Testing Liver-Specific Properties ofCompounds of General Formula (I)

Materials & Methods

Animal Model and Treatment: Ob/Ob Mice

Male ob/ob mice (8 weeks of age) were purchased from Charles River(L'Arbresle, France) and were kept on a 12-hour light/dark cycle at aconstant temperature of 20±3° C. After a 1 week acclimation, mice wereseparated in groups of 8 animals selected such that the distribution oftheir body weight and their 6 hours fasting glycemia determined beforethe experiment were uniform. Animals were fed a standard chow diet (R03,SAFE) and treated during 26 days with the compounds of interest.Compounds, including the Compound 29 of WO2004005233 (Cpd 29, at 10 or30 mg/kg/day). Fenofibrate (100 mg/kg/day) and Rosiglitazone (10mg/kg/day) were administered daily by gavage. Control animals weretreated with vehicle only (Carboxymethlycellulose 1%+Tween-80 0.1%).Animals had access to food and water ad libitum.

Animal Model and Treatment: Study in hApoE2 Knock-in Mice

Female hApoE2 knock-in (KI) transgenic mice (Sullivan et al., 1998)(4weeks of age). Mice were kept on a 12-hour light/dark cycle at aconstant temperature of 20±3° C. After a 1 week acclimation, mice wereseparated in groups of 7-10 animals selected such as the distribution oftheir body weight and plasmatic lipid levels determined before theexperiment were uniform. Animals were fed a Western diet (20% saturatedfat and 0.2% cholesterol, Harlan Teklad TD88137) at weaning and during12 weeks. Compounds of interest (Cpd 29 at 0.3 mg/kg/day and Fenofibrateat 100 mg/kg/day) were incorporated in the Western diet (SAFE, Augy,France) and administrated to mice during 12 weeks. Control animalsreceived Western diet only. Animals had access to food and water adlibitum.

Animal Model and Treatment: Studies in hApoE2 KI and hApoE2 KI PPARalphaKO Mice

Female hApoE2 knock-in (KI) and hApoE2 KI/PPARalpha knock-out (KO)age-matched transgenic mice (8 to 25 weeks of age for the firstexperiment and 10 to 14 weeks of age for the second experiment. hApoE2KI/PPARalpha KO mice were generated by crossing of homozygous hApoE2 KImice (Sullivan P et al., 1998) and homozygous PPARalpha deficient mice(Lee et al., 1995). Mice were kept on a 12-hour light/dark cycle at aconstant temperature of 20±3° C. After a 1 week acclimation, mice wereseparated in groups of 4-6 animals selected such that the distributionof their age, body weight and plasmatic lipid levels determined beforethe experiment were uniform. Animals were fed a Western diet (20%saturated fat and 0.2% cholesterol, Harlan Teklad TD88137) during 2weeks in the first study, that involved the daily administration of Cpd29 (at 30 mg/kg/day by oral gavage), and during 6 weeks in the secondstudy, that involved the daily administration of Compound 1 ofWO2007147879 (Cpd1, at 30 mg/kg/day by oral gavage). Control animalswere treated with vehicle only (Carboxymethlycellulose 1%+Tween-800.1%). Animals had access to food and water ad libitum.

Preparation of Biological Samples Obtained from Animal Models

At the end of the studies, the animals were weighed and sacrificed underanesthesia. Blood was collected from the retro-orbital sinus; plasma wasobtained by centrifugation (4 000 rpm, at 4° C. for 15 min) andsubsequently frozen and stored at −20° C. Tissues and livers wereisolated and snap-frozen in liquid nitrogen and stored at −80° C. forsubsequent analysis (gene expression and biochemistry) or fixed in 4%paraformaldehyde for histology.

Plasma Analysis

Alanine aminotransferase levels were determined in plasma using the RXDaytona™ automatic analyzer (Randox) and the appropriate dosage kit(Randox, cat#AL 3801).

Gene Expression Analysis

Total RNA was isolated from frozen livers using the NucleoSpin™ 96 RNAkit (Macherey Nagel), according to the manufacturer's instructions.Reverse transcription was performed on 1 μg of total RNA by action of 1μl of MMLV-RT enzyme (Invitrogen) during 1 hour at 37° C. in a totalvolume of 30 μl. The reaction conditions were 1× buffer (Invitrogen),1.5 mM DTT (Invitrogen), 0.18 mM dNTPs (Promega), 200 ng pdN6(Amersham), 30 U RNase inhibitor (Promega). Quantitative PCR was thencarried out using the MyiQ Single-Color Real-Time PCR Detection System(Biorad). Briefly PCR reactions were performed in 96 well plates on 5 μlof diluted reverse transcription mix using the iQ SYBR Green Supermixkit. The reaction conditions were: 25 μl of volume reaction, 3 mM ofMgCl2, and 0.5 μl of each reverse and forward primer solutions (10pMol), Tm of 60° C. The pairs of primers that have been designed for thespecific amplification of each target genes are summarized in Table 1.

TABLE 1 Reverse primer  Forward primer  Genes (5′-3′) (5′-3′) 36B4GGGAAGGTGTAATCCGTCTC CATGCTCAACATCTCCCCCT CACAG (SEQ ID NO: 1)TCTCC (SEQ ID NO: 2) TNF AGGTACAACCCATCGGCTGG CGTCGTAGCAAACCACCAAG alpha(SEQ ID NO: 3) TG (SEQ ID NO: 4) TGF TGGTTGTAGAGGGCAAGGACTTGCTTCAGCTCCACAGAGA beta (SEQ ID NO: 5) (SEQ ID NO: 6) CCL5CACACTTGGCGGTTCCTTCG CCCTCACCATCATCCTCACT (SEQ ID NO: 7)GC (SEQ ID NO: 8 Col1a1 GCCAGGAGAACCAGCAGAG AGGCGAACAAGGTGACAGAG(SEQ ID NO: 9) (SEQ ID NO: 10)

The quantity of fluorescence emitted is directly proportional to thequantity of complementary DNA present at the start of the reaction andamplified during the PCR. The relative levels of expression weredetermined using the standard curve for each transcript. The resultswere then normalized in regard to the signals obtained with the 36B4control (a reference transcript for hepatic gene expression). Theinduction factor, i.e. the ratio between the relative signal induced bythe compound according to the invention and the average of the valuesrelating to the control group, was then calculated for each sample. Thehigher this factor, the more the compound promotes target geneexpression. The final result is depicted as the average of the inductionvalues in each experimental group.

Histological Analysis of Liver

Formalin-fixed liver tissue was processed, and 5-μm-thick paraffinsections were stained with Hematoxylin and Eosin. The histologicalanalysis of stained liver sections was carried out in blind conditionsto quantify liver steatosis and liver intralobular inflammation. Liversteatosis was scored from 0 to 3 as follows: 0 (very slightly affected),1 (slightly affected), 2 (moderately affected), 3 (highly affected).Liver intralobular inflammation was also scored depending on the numberof inflammatory foci counted by field of observation as follows: 0 (<1focus/field), 1 (1 to 2 foci/field), 2 (2 to 4 foci/field) 3 (more than4 foci/field).

Hepatic Lipid Analysis

Approximately 100 mg of frozen liver tissue were homogenized with atissue homogenizer (Precellys®24, Bertin Technologies, France) in 150 mMNaCl buffer, containing 15.4 mM NaN3. Lipid fractions in homogenateswere extracted with chloroform-methanol (2:1, v/v) followed bymeasurement of the total cholesterol (using the Cholesterol RTU™ 61218kit, Biomerieux, France) and true triglycerides (TR0100 kit,Sigma-Aldrich).

Results & Conclusions

Several animal models are disclosed in the literature as reflecting theetiology, disease progression, and the pathology of human liverdiseases. However, these models do not always display the range ofhisto-pathological and patho-physiological features associated withspecific liver diseases. As recently reviewed (Fan J and Qiao L, 2009),this is particularly evident in the case of NAFLD or NASH, whereingenetic (in transgenic mice), nutritional (in rats or mice), or mixedmodels have been established.

NASH is characterized by pathological alterations of liver ranging fromsteatosis and liver inflammation to liver degeneration, fibrosis andcirrhosis. The pathogenesis of NASH remains poorly understood. It is acomponent of the metabolic syndrome and therefore frequently associatedwith hyperlipidemia. Different transgenic animal models were used tocharacterize the effects of exemplary compounds of the General Formula(I), and more specifically of General formula (I) and General Formula(IV): the insulin-resistant, leptin deficient ob/ob mice and thedyslipidemic hApoE2 knock-in mice (the latter one, with or without afurther genomic modification consisting in the inactivation of PPARalphagene).

Leptin deficient ob/ob mice are obese, dyslipidemic, insulin resistantand develop hepatic injury and steatosis. Hepatic steatosis isrelatively asymptomatic but individuals with this disorder are atgreater risk for developing NASH. This first protocol was designed toanalyze the effects of the Cpd 29 and of the reference compoundsFenofibrate and Rosiglitazone on the early stages of NASH. i.e.inflammation in the steatosic liver of ob/ob mice. In ob/ob mice, 26days of treatment with the Rosiglitazone induced the increase of thehepatic expression of TNFalpha in the ob/ob mice while no major changein the expression of this cytokine was observed in animals treated withthe Fenofibrate. On the contrary, the administration of Cpd 29 inhibitedthe expression of this cytokine in a dose-response manner (FIG. 2A).Following the same treatment, the hepatic expression level of TGFbetawas equivalent in all control and reference groups (control, Fenofibrateand Rosiglitazone). Again, the administration of Cpd 29 inhibited theexpression of this growth factor in a dose-response manner, an effectthat is more statistically relevant when Cpd 29 was administered at 30mg/kg/day (FIG. 2B).

Plasma ALAT was measured as a surrogate marker for assessing liverinjury in these ob/ob mice after the 26 days of treatment with thedifferent compounds. When the level of plasma ALAT is compared witheither the control group or the Fenofibrate-treated group, the group ofmice that were treated with Rosiglitazone exhibited a significantincrease of their plasmatic levels of ALAT. On the contrary, theadministration of Cpd 29 at 30 mg/kg/day induced a statisticallysignificant decrease of plasmatic levels of ALAT (FIG. 2C).

Another in vivo model was used in order to study the effects of Cpd 29and of the reference compound Fenofibrate on physiological parametersnormally being considered as relevant for assessing NASH. In “humanized”ApoE2 knock-in mice (referred as hApoE2 KI) the human ApoE2 allelereplaces the murine apoe gene, so that these mice express human ApoE2(hApoE2) under the control of the endogenous promoter sequences atphysiological levels. However, hApoE2 has a markedly reduced affinityfor the LDL receptor, leading to a plasma lipoprotein profile resemblinghuman type III hyperlipoproteinemia (Sullivan et al., 1998). Similar tohumans, hApoE2 KI mice are responsive to lipid-lowering drugs such asfibrates (ligands for PPARα). This class of drugs has been shown toreverse steatohepatitis in mice (Shiri-Sverdlov R et al., 2006) and thusthis model can allow evaluating liver-specific anti-inflammatory andanti-fibrotic effects of compounds of General Formula (I). Inparticular, elevated TNFα levels are related to liver inflammation,necrosis and fibrosis typical of NASH (Larter et al., 2008). TGFβ is apeptide found in many cell types that regulates wound healing andapoptosis. The isoform found in hepatic cells, TGFβ1, has been found inmany models of hepatic fibrosis and levels increase in chronic activehepatitis and fibrotic alcoholic liver disease (Nan et al., 2009).

The different hApoE2 KI mice were treated during 12 weeks while fed aWestern diet. In this model, Cpd 29 inhibited the hepatic expression ofgene that are relevant for liver inflammation (TNFα, CCL5, TGFβ; FIG.3A, 3B, 3C respectively) with an efficacy similar (if not superior) toFenofibrate that was administered at an higher dose.

However, the mice group treated with Cpd 29 showed a statisticallysignificant inhibition of the expression of genes like those forspecific collagen chains that are involved in liver fibrosis(Basaranoglu et al., 2010), and in particular Col1a1 (FIG. 3D). Such aneffect on collagen genes was not observed in the mice group treated withFenofibrate. These results demonstrate that Cpd29 displaysanti-inflammatory and anti-fibrotic properties in an in vivo model ofNASH.

Exemplary compounds of the General Formula (I) were tested in vivo in ahigh fat diet mice model. hApoE2 KI and hApoE2 KI/PPARalpha KO(“humanized” ApoE2 knock-in mice deficient for the mPPARalpha gene) werefed a Western diet and daily treated with the Cpd 29 at 30 mg/kg/dayduring 2 weeks. At the end of the protocol, liver steatosis andintra-lobular inflammation were evaluated in the control and treatedmice by means of histological analysis and specific scores.

This study demonstrated that the treatment with Cpd 29 inhibits thedevelopment of both liver steatosis and liver inflammation that isinduced by the diet in hApoE2 KI mice and, even more rapidly in hApoE2KI/PPARalpha KO mice, wherein it is evident an acceleration of the liverdisorder due to the lack of PPARalpha (Table 2 and Table 3).

TABLE 2 Liver hApoE2 KI hApoE2 KI/PPARalpha KO Steatosis Cpd 29 Cpd 29(score) Vehicle (30 mg/kg/day) Vehicle (30 mg/kg/day) 0 17% 83%  0%  0%1 50% 17% 33% 33% 2 33%  0% 33% 50% 3  0%  0% 33% 17%

TABLE 3 Liver hApoE2 KI hApoE2 KI/PPARalpha KO Inflammation Cpd 29 Cpd29 (score) Vehicle (30 mg/kg/day) Vehicle (30 mg/kg/day) 0 50% 100%  17%67% 1 50% 0% 33% 17% 2  0% 0% 33% 17% 3  0% 0% 17%  0%

In another study, the liver-specific anti-inflammatory and anti-fibroticproperties effects of Cpd 29 and Compound 1 of WO2007/147879 (Cpd 1)were evaluated in the hApoE2 KI/PPARalpha KO mice that were fed aWestern diet and daily treated with the selected compounds during 6weeks.

Both Cpd 29 and Cpd 1 inhibited the liver expression of TNFα, TGFβ andcollagen in hApoE2 KI/PPARalpha KO mice (FIGS. 4A, 4B, and 4C,respectively), confirming the liver-specific (mainlyPPARalpha-independent), anti-inflammatory, and anti-fibrotic propertiesof these compounds in a relevant in vivo model for NASH. The hepaticlipids analysis further revealed that both Cpd 29 and Cpd 1 preventedthe triglycerides accumulation in the liver of hApoE2 KI/PPARalpha KOmice (FIG. 4D).

Taken all together, those results highlighted the liver-specificanti-inflammatory, anti-steatosic and anti-fibrotic properties of theCompound 29 of WO2004/005233 (comprised in General Formula I and II) andthe Compound 1 of WO2007/147879 (comprised in General Formula (I) and(IV)) in vivo.

Additional model for testing the compounds of General Formula (I) arenutritional animal models of NASH such as the methionine- andcholine-deficient (MCD) model which is based on a diet containing highsucrose and fat but lacks two components, methionine and choline thatare essential factors for liver metabolism. Mice or rats fed on thisdiet rapidly develop hepatic inflammation, which further evolves intosteatosis, necrotic inflammation, fibrosis, and oxidative stress. Thisapproach has been used to show the potential therapeutic effects onliver steatosis, fibrosis, oxidative stress, and/or inflammation thatare associated to the administration of compounds such as Rosiglitazone(Tahan V., et al. 2007), the pan-caspase inhibitor VX-166 (Witek R etal., 2009), Zeaxantin (Chamberlain S et al., 2009), Telmisartan (Kudo Het al., 2009) or Wy-14,643 (Ip E et al., 2004).

The compounds of General Formula (I) can be tested in an MCD modelestablished in Sprague Dawley rats (8 weeks old) or C57B16 mice that arefed with the methionine- and choline-deficient diet during 4 to 12weeks. Treatment with the compounds of interest, including compoundsthat are chosen as negative or positive control, are then administereddaily at different doses to groups of ten or more animals by gavageduring the following 4 to 12 weeks. Several types of measurements can beperformed before, during, or at the end of the treatment, with orwithout sacrificing the animals. Biochemical dosages (aspartateaminotransferase and alanine aminotransferase activities, totalbilirubin, alkaline phosphatase, LDL/HDL-cholesterol, serum hyaluronate,hepatic triglyceride and plasmatic triglyceride) and histomorphometricanalysis (for determining the liver area presenting fibrosis and/orsteatosis) are the more relevant measurements. The dosage ofinflammatory markers (such as Interleukins-1a, -1b, -2, -4, -6, -10.Interferon gamma, or TNFalpha) and/or of the expression of relevantgenes (such as type I collagen or liver-specific chemokine receptors)can be also evaluated.

Alternatively, animal models based on the chemically-induced hepaticfibrosis can be used for studying the antifibrotic effect of thecompounds of General Formula (I). For example the administration ofthioacetamide (TAA) or carbon tetrachloride (CCL4) induces an increasein reactive oxygen species (ROS) promoting lipid peroxidation, hepaticstellate cell proliferation and collagen hyperproduction, leading tochronic liver injury and fibrosis in rats. This approach has been usedto show the positive effect on liver fibrosis, oxidative stress, and/orinflammation with compounds such as Curcumin (Fu Y et al., 2008) orPioglitazone (Yuan G et al., 2004).

The compounds of General Formula (I) can be tested in an CCL4 model thatis established in Sprague Dawley rats (8 weeks old) which receiveincreasing doses of CCL4 intraperitoneally diluted in liquid paraffin(of 50%) every five days for 4 to 12 weeks. Phenobarbital can be alsoadministered starting from 10 days before the first dose of CCL4 inorder to potentiate the model. Treatment with the compounds of interest,including compounds that are chosen as negative or positive control, arethen administered daily at different doses (comprised between 0.01 and100 mg/kg/day) to groups of ten or more rats by gavage during thefollowing 4 to 12 weeks. As in the MCD model, several types ofmeasurements can be performed before, during, or at the end of thetreatment, with or without sacrificing the rats, for evaluating theefficacy of the treatment on the basis of biochemical dosages andhistomorphometric analysis, in association to hemodynamic indexes andthe dosage of inflammatory markers and/or of the expression of relevantgenes.

The animal models described above allow comparing liver-specificactivities of compounds of General Formula (I) among them and withcompounds already known as having liver-specific (and in particularNAFLD-/NASH-specific) therapeutic properties. In particular, the datashown in this Example suggest the superiority of compounds of GeneralFormula (I) when compared to reference compounds.

Example 3 In Vitro/Ex Vivo Models for Testing Liver-Specific Propertiesof Compounds of General Formula (I)

Some in vitro/ex vivo models have been established for screeningcompounds that can have a positive effect on liver fibrosis, oxidativestress, and/or liver inflammation. In fact, a key event in liverfibrosis is the activation of hepatic stellate cells (HSC). Afterhepatocyte damage, this cell type becomes activated and starts toproliferate (Sato M et al., 2003). Activated HSCs (for example, rat orhuman HSC isolated from livers or rat HSC-T6 cell line) can be activatedand produce excessive amounts of extracellular matrix compounds andinhibitors of matrix degradation. This approach has been used to showthe positive effect of compounds such as curcumin (Xu et al., 2003),thiazolidinediones (Miyahara T et al., 2000), or 17beta-estradiol (Liu Qet al., 2004).

The in vitro/ex vivo models described above allow comparingliver-specific activities of compounds of General Formula (I) among themand with compounds known as having liver-specific (and in particularNAFLD-/NASH-specific) therapeutic properties.

Example 4 Effects of Compounds of General Formula (I) on PathwaysResponsible for Fibrosis

The inventors have also conducted experiments with other compounds ofGeneral Formula (I) showing that said compounds are able to act onpathways responsible for fibrosis, which is a key feature of a number ofliver disorders, in particular NAFLD, NASH and cirrhosis. Theseexperiments are summarized in table 4.

Materials & Methods

Compounds were dissolved in dimethyl sulfoxide (DMSO, Fluka cat#41640)

hHSC Culture and Treatment Conditions

The human primary hepatic stellate cells (hHSC) (ScienCell) werecultured in STeCM medium (ScienCell cat#5301) that was supplemented with2% fetal bovine serum (FBS, ScienCell cat#0010), 1%penicillin/streptomycine (ScienCell cat#0503) and stellate cell growthsupplement (SteCGS; ScienCell cat#5352). Culture plastics were coatedwith Poly-L Lysine (Sigma cat#P4707).

Determination of PDGF-Induced Proliferation

hHSC were plated at a density of 1.2×10⁴ cells/well into 96-well platesand were cultured overnight at 37° C. and 5% CO₂, followed by washing ofcells with PBS (Invitrogen cat#14190) and replacing the growth mediumwith a serum-free and SteCGS-free medium for an additional 24 hours. ForPDGF-induced proliferation assay, cells were pre-treated with a compoundof General Formula (I) for 1 hour before the addition of PDGF-BB (10ng/ml; R&D Systems cat#520-BB). Treatments were then continued foradditional 20 hours. Cell proliferation was measured bybromodeoxyuridine (BrdU) incorporation using a BrdU labeling anddetection kit (Roche cat#11647229001). BrdU labeling solution was addedto cells, followed by incubation for another 4 hours before fixation,addition of nucleases, addition of anti-BrdU-POD and peroxidasesubstrate. The absorbance at 405 nm (with a reference wavelength at 690nm) was measured using an ELISA plate reader (Tecan).

HSC Activation with TGF-β1

The human primary hepatic stellate cells (hHSC) (ScienCell) werecultured under standard conditions, as described above. For experimentsto determine gene expression patterns, hHSC were plated at a density of7×10⁴ cells/well into 24-well plates and were cultured overnight. Nextday, culture medium was replaced by serum-free and SteCGS-free mediumfor an additional 16 hours. Cells were treated with a compound ofGeneral Formula (I) in addition to TGFβ1 (1 ng/ml) in a serum-free andSteCGS-free medium for 24 hours.

Gene Expression

Total RNA was isolated using Nucleospin® 96 RNA kit (Macherey Nagel)following manufacturer's instructions. 100 ng of total RNA were reversetranscribed in cDNA using M-MLV RT (Moloney Murine Leukemia VirusReverse Transcriptase) (Invitrogen cat#28025) in presence of RT buffer1× (Invitrogen), 1 mM DTT (Invitrogen), 0.18 mM dNTPs (Promega), 200 ngpdN6 (Amersham) and 30 U of RNase inhibitor (Promega).

Quantitative PCR was then carried out using the CFX96 TOUCH™ Real-TimePCR Detection System (Biorad). Briefly, PCR reactions were performed in96 well plates on 5 μl of 5× diluted reverse transcription mix using theiQ SYBR Green Supermix kit. The experimental conditions were: 25 μl ofvolume reaction. 3 mM of MgCl₂, and 0.5 μl each of reverse and forwardprimers (10 pMol).

TABLE 4 Primer name Sequence (5′->3′) αSMA forward ACTGCCTTGGTGTGTGACAA(SEQ ID NO: 11) αSMA reverse TGGTGATGATGCCATGTTCT (SEQ ID NO: 12)Col1α1 forward AATGGTGCTCCTGGTATTGC (SEQ ID NO: 13) Col1α1 reverseACCAGGTTCACCGCTGTTAC (SEQ ID NO: 14) Col4α1 forward GTTGGTCTACCGGGACTCAA(SEQ ID NO: 15) Col4α1 reverse GTTCACCTCTGATCCCCTGA (SEQ ID NO: 16)

Expression levels were normalized using the expression of 36B4 gene asreference (sequences SEQ ID NO:1 and SEQ ID NO:2 as shown above).

For each gene, the standard curves were drawn by selecting the bestpoints (at least three points) in order to have PCR reaction efficiencyclose to 100% and a correlation coefficient close to 1. Expressionlevels were determined using the standard curve equation for both thehousekeeping gene and the target gene (taking into account the specificPCR efficiency of each target gene).

TABLE 5 Inhibition of the PDGF- BB-induced hHSC proliferation byInhibition of the TGFβ1-induced hHSC activation compounds of formula bycompounds of formula (I) at 10 μM (I) at 10 μM αSMA Col1α1 Col4α1 BrdUIncorporation Cpd 74 −97% ***    −82% *** −85% *** −127% ***US20070032543 Cpd 11 −92% ***   −66% ** −78% *** −6%  WO2005073184 Cpd56 −87% ***   −62% ** −66% *** −127% *** US20070032543 Cpd 72 −74% ***−37% −51% *** −126% *** US20070032543 Cpd 33 −71% ***  −58% * −73% ***35%  WO2004OO5233 Cpd 84 −64% ***   −60% ** −64% *** −33%  US20070032543 Cpd 54 −57% *** −27% −40% *** 12%  US20070032543 Cpd 80−55% ***   −64% ** −66% *** −136% *** US20070032543 Cpd 33 −49% *** −37%12%   −127% *** WO2005073184 Cpd 17 −30% *** −29% −56% *** 6% WO2004OO5233 Cpd 41 −2%   −28% −32% **  −34%   WO2004OO5233 Cpd 58 5%    55% * −3%   −5%  US20070032543

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We claim:
 1. A method for the treatment of a non-alcoholic fatty liverdisease, comprising administering to a patient having non-alcoholicfatty liver disease a pharmaceutical composition comprising: atherapeutically effective amount of1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-oneor a pharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable excipient.
 2. The method of claim 1, wherein the compositionis administered to the patient once daily.
 3. The method of claim 1,wherein the composition is administered to the patient orally.
 4. Themethod of claim 1, wherein the composition is a tablet.
 5. The method ofclaim 1, wherein the non-alcoholic fatty liver disease is non-alcoholicsteatohepatitis.
 6. A method for the treatment of non-alcoholicsteatohepatitis, comprising orally administering once daily to a patienthaving non-alcoholic steatohepatitis a tablet comprising: atherapeutically effective amount of1-[4-methylthiophenyl]-3-[3,5-dimethyl-4-carboxydimethylmethyloxyphenyl]prop-2-en-1-oneor a pharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable excipient.